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Figure 5 Both tumor models may play a role in the maintenance of a tumor. Initially, tumor growth is assured with a specific CSC (CSC 1). With tumor progression, another CSC (CSC 2) may arise due to the clonal selection. The development of a new more aggressive CSC may result from the acquisition of an additional mutation or epigenetic modification.
Researchers at Stanford’s Ludwig Center for Cancer Stem Cell Research and Medicine, for instance, discovered why stem cells, including cancer stem cells, are resistant to the ionizing radiation used in many cancer treatments. This understanding may help researchers find compounds that make CSCs vulnerable to radiation damage. Another example concerns immune therapies in which the body’s immune system is trained to attack cancer cells. Some of these therapies failed in clinical trials of skin cancer, and Stanford researchers demonstrated why: the targets that the immune system were trained to attack belonged not to the CSCs but to slightly different daughter cells. The immune therapies seemed effective at first as they attacked the daughter cells, but they left the skin cancer stem cells untouched and therefore it could not cure the cancer.
The first conclusive evidence for CSCs was published in 1997 in Nature Medicine. Bonnet and Dick isolated a subpopulation of leukemic cells that expressed a specific surface marker CD34, but lacked the CD38 marker. The authors established that the CD34+/CD38– subpopulation is capable of initiating tumors in NOD/SCID mice that are histologically similar to the donor. The first evidence of a solid tumor cancer stem–like cell followed in 2002 with the discovery of a clonogenic, sphere-forming cell isolated and characterized from human brain gliomas. Some researchers favor the theory that the CSC is generated by a mutation in stem cell niche populations during development. These developing stem populations are mutated and then expanded such that the mutation is shared by many of the daughter cells of the mutated stem cell.
Another theory associates adult stem cells with the formation of tumors, most often in tissues with a high rate of cell turnover (such as the skin or gut). In these tissues, stem cells are thought to be responsible for tumor formation as a result of the frequent cell divisions of these stem cells (compared to most adult stem cells) in conjunction with the extremely long life span of adult stem cells. This combination creates the ideal set of circumstances for mutations to accumulate; accumulation of mutations is the primary factor that starts a cancer. Another theory is that the mutated cells de-differentiate into stem cells.
Isolation of Cancer Stem Cells
CSCs are commonly identified and enriched using strategies for identifying normal stem cells, including fluorescence-activated cell sorting (FACS), with antibodies directed at cell-surface markers, and functional approaches including side population analysis or Aldefluor assay (an assay that detects stem and progenitor cells based on their expression of the enzyme aldehyde dehydrogenase, rather than cell surface phenotype). The CSC-enriched population purified by these approaches is then implanted, at various cell doses, in immune-deficient mice to assess tumor development capacity. This in vivo assay is called limiting dilution assay. The tumor cell subsets that can initiate tumor development at low cell numbers are further tested for self-renewal capacity in serial tumor studies. CSCs can also be identified by efflux of incorporated Hoechst dyes via multidrug resistance (MDR) and ATP-binding cassette (ABC). The use of Oct4 as a marker for stemness of embryonic stem cells is well established. Oct4 expression has been demonstrated in several adult normal and cancer tissues, in adult human and canine tissues, in human cancer cell lines, and in normal adult human stem cells.
Another in vitro approach for identification of cell subsets enriched with cancer stem cells is sphere-forming assays. Many normal stem cells are capable, under special culture conditions, of forming three-dimensional spheres, which can differentiate into multiple cell types. As with normal stem cells, the CSCs isolated from brain or prostate tumors also have the ability to form anchorage-independent spheres.
Data over recent years have indicated the existence of CSCs in various solid tumors. For isolating CSCs from solid and hematological tumors, markers specific for normal stem cells of the same organ are commonly used. A number of cell surface markers have proven useful for isolation of subsets enriched for cancer stem cells, including CD133 (also known as PROM1), CD44, CD24, EpCAM (epithelial cell adhesion molecule, also known as epithelial specific antigen, ESA), THY1, and ATP-binding cassette B5 (ABCB5).
CD133 (prominin 1)
is a five-transmembrane domain glycoprotein expressed on CD34+ stem and progenitor cells, in endothelial precursors and fetal neural stem cells. It has been detected using its glycosylated epitope known as AC133.
EpCAM (epithelial cell adhesion molecule, ESA, TROP1)
is a hemophilic Ca2+-independent cell adhesion molecule expressed on the basolateral surface of most epithelial cells.
CD90 (THY1)
is a glycosylphosphatidylinositol glycoprotein anchored in the plasma membrane and involved in signal transduction. It may also mediate adhesion between thymocytes and thymic stroma.
CD44 (PGP1)
is an adhesion molecule that has pleiotropic roles in cell signaling, migration, and homing. It has multiple isoforms, including CD44H, which exhibits high affinity for hyaluronate, and CD44V, which has metastatic properties.
CD24 (HSA)
is a glycosylated glycosylphosphatidylinositol-anchored adhesion molecule, which has a co-stimulatory role in B and T cells.
ALDH
is a ubiquitous aldehyde dehydrogenase family of enzymes, which catalyzes the oxidation of aromatic aldehydes to carboxyl acids. For instance, it has a role in conversion of retinol to retinoic acid, which is essential for survival.
The first solid malignancy from which CSCs were identified was breast cancer and these CSCs are the most intensely studied. Breast cancer stem cells have been enriched in CD44+CD24–/low, SP, ALDH+ subpopulations. However, recent evidence indicates that breast CSCs are very diverse. There is also evidence that CSC marker expression in breast cancer cells are heterogeneous and that there exist many subsets of breast CSC. Both CD44+CD24– and CD44+CD24+ cell populations are tumor-initiating cells; however, CSCs are most highly enriched using the marker profile CD44+CD49fhiCD133/2hi.
CSCs have been reported in many brain tumors. Stem-like tumor cells have been identified using cell surface markers including CD133, SSEA-1 (stage-specific embryonic antigen-1), EGFR, and CD44. However, there is uncertainty about the use of CD133 for identification of brain tumor stem–like cells because tumorigenic cells are found in both CD133+ and CD133– cells in some gliomas, and some CD133+ brain tumor cells may not possess tumor-initiating capacity. CSCs have also been reported in human colon cancer.
Metastasis is the major cause of tumor lethality in patients, but not every cell in the tumor has the ability to metastasize. This potential depends on factors that determine growth, ability to form new blood vessels, invasion of surrounding structures, and other processes of tumor cells. In many epithelial tumors, the epithelial-mesenchymal transition (EMT) is considered a crucial event in the metastatic process. EMT and the reverse transition from mesenchymal to an epithelial phenotype (MET) are involved in embryonic development, which involves disruption of epithelial cell homeostasis and the acquisition of a migratory mesenchymal phenotype. The EMT appears to be controlled by pathways such as WNT and transforming growth factor β (TGF-β) pathway. The
